Protein S is a vitamin K-dependent plasmatic protein known since 1977. Initially, no function was attributed to protein S and only recently has its role in manifesting the anticoagulant activity of activated protein C been proposed.
Protein C is a protein of the prothrombinic complex the function of which is to inhibit the enzymatic cascade of plasmatic coagulation at Factor V and Factor VIII level.
Clinical studies have also shown that patients congenitally deficient in protein C are prone to frequent thrombosis. In vitro studies have shown that protein S acts as cofactor for activated protein C. These studies have therefore suggested that protein S is essential for manifesting the anticoagulant effects of activated protein C.
As protein S is necessary for the optimum expression of the anticoagulant activity of activated protein C, it has been suggested that a deficiency of protein S can make an individual prone to illnesses of a thrombotic nature.
The determination of protein S.in the plasma is therefore of great interest, in that its greater or lesser deficiency constitutes a thrombosis risk factor.
In human plasma, protein S forms a equimolar complex with another plasmatic protein, namely C4b-BP.
At equilibrium under physiological conditions protein S exists in two forms, namely protein S bound to C4b-BP (=60%) and free protein S. Free protein S is the only form to exhibit the biological function of cofactor for activated protein C.
The methods proposed up to now for determining the biological activity of protein S basically comprise this series of steps:
mixing the sampLe to be tested, either w or suitably diluted, with plasma deficient in protein S;
adding an optimum quantity of activated protein C or PROTAC, to activate the protein C of the deficient plasma;
adding an optimum quantity of phospholipids and, if it is necessary, of activated factor;
adding CaCl.sub.2 as initiator for the coagulation reaction;
evaluating the activity of protein S in the sample by expressing it as a percentage calculated from a calibration curve which has been constructed using a plasma pool from healthy individuals, for which the protein S activity is assigned the value 100%.
Functional tests for measuring the activity of protein S in plasma are described for example in "Thrombosis Research" vol. 48, 427-437, 1987 (P. van de Waart et al.) and idem vol. 49, 241-251, 1988 (K. Suzuki et al.); in "Blood" vol. 67, 406-410, 1986 and in "J. Clin. Invest." vol. 81, 1445-1454, 1988 and vol. 74, 2082-2086, 1984.
These tests are therefore based on the capacity of protein S to serve as cofactor for the anticoagulant effects of activated protein C. The prolongation of the coagulation time induced by activated protein C depends on the content of protein S in the plasma of the sample.
The object of the invention is to provide a method for determining the functional activity of protein S which can be carried out using not excessively complicated techniques in normal analysis laboratories at acceptable cost, and with greater accuracy and repeatability than with the previously proposed methods.